Our long-term objective is to elucidate the mechanisms of renin secretion. Increased extracellular calcium (Ca2+) decreases renin release in vitro by increasing juxtaglomerular (JG) cell intracellular Ca2+ and inhibiting adenylyl cyclase V (AC-V). How extracellular Ca2+ decreases rennin secretion in vivo and what initiates these changes in Ca2+ is unknown. Our hypothesis is that increased distal NaCI delivery increases Ca2+ in the renal cortical interstitium via paracellin-1 which stimulates the calcium-sensing receptor (CaR), inhibiting AC-V activity, leading to a decrease in renin secretion. AIM 1: Hypothesis: Increasing thick ascending limb NaCI transport increases cortical interstitial [Ca2+] via paracellin-1 and decreases renin secretion. We will measure changes in renal cortical interstitial Ca2+ using in situ microdialysis and changes in the renin secretory rate (RSR) in response to changes in NaCI delivery. We will measure interstitial Ca2+ and RSR after decreasing the expression of paracellin-1, a tight-junction protein responsible for paracellular Ca2+ reabsorption in the thick ascending limb, using RNA interference. We will measure changes in interstitial Ca2+ and RSR in response to the blockade of thick ascending limb NaCI reabsorption with furosemide. AIM 2: Hypothesis: In vivo stimulation of the CaR via cortical interstitial [Ca2+] decreases renin secretion. We will measure RSR while using calcimimetics to sensitize or the CaR in rats. We will measure changes in RSR to CaR activation in the presence of decreased interstitial [Ca2+] due to paracellin-1 knockdown. We will measure changes in RSR to CaR desensitization with calcilytics. We will measure RSR in the presence of increased interstitial [Ca2+] and CaR desensitization with calcilytics. AIM 3: Hypothesis: In vivo AC-V inhibition decreases renin secretion in the presence of decreased cortical interstitial [Ca2+]. We will measure basal RSR in response to the AC-V specific inhibitor NKY80. We will knockdown paracellin-1 expression in rats using siRNA technology and will measure interstitial [Ca2+] and RSR in the presence of the AC-V specific inhibitor, NKY80. We will knockdown AC-V expression in vivo using RNA interference, and measure interstitial [Ca2+] and RSR in response to furosemide. The renin-angiotensin system (RAS) is involved in almost every form of hypertension. Inhibition of the RAS is a mainstay of anti-hypertensive therapy. Understanding the function and regulation of the RAS will allow for the development of novel therapeutics to lessen the impact of high blood pressure.